Regulation of mTORC1 Signaling by pH

BACKGROUND: Acidification of the cytoplasm and the extracellular environment is associated with many physiological and pathological conditions, such as intense exercise, hypoxia and tumourigenesis. Acidification affects important cellular functions including protein synthesis, growth, and proliferation. Many of these vital functions are controlled by mTORC1, a master regulator protein kinase that is activated by various growth-stimulating signals and inactivated by starvation conditions. Whether mTORC1 can also respond to changes in extracellular or cytoplasmic pH and play a role in limiting anabolic processes in acidic conditions is not known. METHODOLOGY/FINDINGS: We examined the effects of acidifying the extracellular medium from pH 7.4 to 6.4 on human breast carcinoma MCF-7 cells and immortalized mouse embryo fibroblasts. Decreasing the extracellular pH caused intracellular acidification and rapid, graded and reversible inhibition of mTORC1, assessed by measuring the phosphorylation of the mTORC1 substrate S6K. Fibroblasts deleted of the tuberous sclerosis complex TSC2 gene, a major negative regulator of mTORC1, were unable to inhibit mTORC1 in acidic extracellular conditions, showing that the TSC1-TSC2 complex is required for this response. Examination of the major upstream pathways converging on the TSC1-TSC2 complex showed that Akt signaling was unaffected by pH but that the Raf/MEK/ERK pathway was inhibited. Inhibition of MEK with drugs caused only modest mTORC1 inhibition, implying that other unidentified pathways also play major roles. CONCLUSIONS: This study reveals a novel role for the TSC1/TSC2 complex and mTORC1 in sensing variations in ambient pH. As a common feature of low tissue perfusion, low glucose availability and high energy expenditure, acidic pH may serve as a signal for mTORC1 to downregulate energy-consuming anabolic processes such as protein synthesis as an adaptive response to metabolically stressful conditions

Niclosamide Prevents the Formation of Large Ubiquitin-Containing Aggregates Caused by Proteasome Inhibition

Protein aggregation is a hallmark of many neurodegenerative diseases and has been linked to the failure to degrade misfolded and damaged proteins. In the cell, aberrant proteins are degraded by the ubiquitin proteasome system that mainly targets short-lived proteins, or by the lysosomes that mostly clear long-lived and poorly soluble proteins. Both systems are interconnected and, in some instances, autophagy can redirect proteasome substrates to the lysosomes.To better understand the interplay between these two systems, we established a neuroblastoma cell population stably expressing the GFP-ubiquitin fusion protein. We show that inhibition of the proteasome leads to the formation of large ubiquitin-containing inclusions accompanied by lower solubility of the ubiquitin conjugates. Strikingly, the formation of the ubiquitin-containing aggregates does not require ectopic expression of disease-specific proteins. Moreover, formation of these focused inclusions caused by proteasome inhibition requires the lysine 63 (K63) of ubiquitin. We then assessed selected compounds that stimulate autophagy and found that the antihelmintic chemical niclosamide prevents large aggregate formation induced by proteasome inhibition, while the prototypical mTORC1 inhibitor rapamycin had no apparent effect. Niclosamide also precludes the accumulation of poly-ubiquitinated proteins and of p62 upon proteasome inhibition. Moreover, niclosamide induces a change in lysosome distribution in the cell that, in the absence of proteasome activity, may favor the uptake into lysosomes of ubiquitinated proteins before they form large aggregates.Our results indicate that proteasome inhibition provokes the formation of large ubiquitin containing aggregates in tissue culture cells, even in the absence of disease specific proteins. Furthermore our study suggests that the autophagy-inducing compound niclosamide may promote the selective clearance of ubiquitinated proteins in the absence of proteasome activity

Nitazoxanide Stimulates Autophagy and Inhibits mTORC1 Signaling and Intracellular Proliferation of Mycobacterium tuberculosis

Tuberculosis, caused by Mycobacterium tuberculosis infection, is a major cause of morbidity and mortality in the world today. M. tuberculosis hijacks the phagosome-lysosome trafficking pathway to escape clearance from infected macrophages. There is increasing evidence that manipulation of autophagy, a regulated catabolic trafficking pathway, can enhance killing of M. tuberculosis. Therefore, pharmacological agents that induce autophagy could be important in combating tuberculosis. We report that the antiprotozoal drug nitazoxanide and its active metabolite tizoxanide strongly stimulate autophagy and inhibit signaling by mTORC1, a major negative regulator of autophagy. Analysis of 16 nitazoxanide analogues reveals similar strict structural requirements for activity in autophagosome induction, EGFP-LC3 processing and mTORC1 inhibition. Nitazoxanide can inhibit M. tuberculosis proliferation in vitro. Here we show that it inhibits M. tuberculosis proliferation more potently in infected human THP-1 cells and peripheral monocytes. We identify the human quinone oxidoreductase NQO1 as a nitazoxanide target and propose, based on experiments with cells expressing NQO1 or not, that NQO1 inhibition is partly responsible for mTORC1 inhibition and enhanced autophagy. The dual action of nitazoxanide on both the bacterium and the host cell response to infection may lead to improved tuberculosis treatment

Induction of Covalently Crosslinked p62 Oligomers with Reduced Binding to Polyubiquitinated Proteins by the Autophagy Inhibitor Verteporfin.

Autophagy is a cellular catabolic process responsible for the degradation of cytoplasmic constituents, including organelles and long-lived proteins, that helps maintain cellular homeostasis and protect against various cellular stresses. Verteporfin is a benzoporphyrin derivative used clinically in photodynamic therapy to treat macular degeneration. Verteporfin was recently found to inhibit autophagosome formation by an unknown mechanism that does not require exposure to light. We report that verteporfin directly targets and modifies p62, a scaffold and adaptor protein that binds both polyubiquitinated proteins destined for degradation and LC3 on autophagosomal membranes. Western blotting experiments revealed that exposure of cells or purified p62 to verteporfin causes the formation of covalently crosslinked p62 oligomers by a mechanism involving low-level singlet oxygen production. Rose bengal, a singlet oxygen producer structurally unrelated to verteporfin, also produced crosslinked p62 oligomers and inhibited autophagosome formation. Co-immunoprecipitation experiments demonstrated that crosslinked p62 oligomers retain their ability to bind to LC3 but show defective binding to polyubiquitinated proteins. Mutations in the p62 PB1 domain that abolish self-oligomerization also abolished crosslinked oligomer formation. Interestingly, small amounts of crosslinked p62 oligomers were detected in untreated cells, and other groups noted the accumulation of p62 forms with reduced SDS-PAGE mobility in cellular and animal models of oxidative stress and aging. These data indicate that p62 is particularly susceptible to oxidative crosslinking and lead us to propose a model whereby oxidized crosslinked p62 oligomers generated rapidly by drugs like verteporfin or over time during the aging process interfere with autophagy

Effect of verteporfin-induced high-MW p62 on its association with polyubiquitinated proteins and LC3.

<p>(A) MCF-7 EGFP-LC3 or (B) BxPC-3 cells were exposed for 4 h to 0.1% DMSO or 10 µM verteporfin in complete medium. p62 was immunoprecipitated and the bound polyubiquitinated proteins were detected using an anti-(Ub)_n antibody. Immunoprecipitation was confirmed by western blot for p62. (C) Densitometry analysis was performed on the images presented in A and B using Quantity One software. (D) Using the same lysates prepared in (A) and (B), EGFP-LC3 was immunoprecipitated and bound p62 was detected in the IP fraction using an anti-GFP antibody. Immunoprecipitation was confirmed by western blot for GFP. Images presented for MCF-7 EGFP-LC3 cells are representative of at least 3 independent experiments. Densitometry was done using images from 2 of those experiments (mean ± S.D., n = 2) where the image quality was suitable for quantification. Images presented for BxPC-3 cells are representative of 2 independent experiments and densitometry was done using images from 1 experiment where the image quality was suitable for quantification.</p

Effect of verteporfin on p62 in cells.

<p>(A) MCF-7 EGFP-LC3 cells were exposed to 0.1% DMSO, 100 nM bafilomycin A1, or 10 µM verteporfin for 8 h in the presence or absence of serum and cell lysates were immunoblotted for p62. β-tubulin was monitored as a loading control. (B) MCF-7 EGFP-LC3 cells were exposed to 0.1% DMSO or 10 µM verteporfin for 4 h. Indicated amounts of each lysate were immunoprecipitated with anti-p62 antibody and analyzed by western blot. (C) MCF-7 EGFP-LC3 cells were exposed to 0.1% DMSO, 10 µM verteporfin, or 100 nM bafilomycin A1 for 4 h in complete medium. The cells were fixed and stained with p62 antibody, and images were acquired by confocal microscopy. Scale bar, 10 µm. (D) MCF-7 EGFP-LC3 cells were exposed to 0.1% DMSO, 100 nM bafilomycin A1, or 10 µM verteporfin for 4 h in complete medium. Cell lysates were collected, quantified, and normalized in the presence or absence of overhead laboratory light as indicated. 0.5 µg of lysate was used to examine p62 levels by western blotting. All images presented are representative of at least 3 independent experiments.</p

Effect of PB1 mutation on p62 crosslinking by verteporfin.

<p>p62^−/− MEF cells expressing GFP-p62 wt or GFP-p62 K7A/D69 or p62^+/+ MEF cells were exposed to 0.1% DMSO or 10 µM verteporfin for 4 h in complete medium. Cell lysates were immunoblotted for p62 and β-tubulin. The image presented is representative of at least 3 independent experiments.</p

Effect of verteporfin on p62 <i>in vitro</i>.

<p>(A) Equal amounts of untreated BxPC-3 cell lysate were exposed to 10 µM verteporfin in the presence or absence of light at 4°C or 37°C for 30 min. (B) p62 was immunoprecipitated from untreated BxPC-3 cells. The immunoprecipitated material was then treated for 30 min in lysis buffer with 10 µM verteporfin in the presence or absence of light at 4°C or 37°C. (C) 100 ng purified GST-p62 was exposed to 10 µM verteporfin for 1 h at 37°C in the absence or presence of light. The above reactions were all immunoblotted for p62. All images presented are representative of at least 3 independent experiments.</p

Effect of different ROS sources on p62 <i>in vitro</i>.

<p>50 ng purified GST-p62 was exposed to different molar ratios of (A) NaOCl or (B) H₂O₂ for 1 h at 37°C, or to different concentrations of (C) peroxynitrite for 5 min at room temperature or (D) DEA/NONOate for 20 min at room temperature. All reactions were immunoblotted for p62. All images presented are representative of at least 3 independent experiments.</p